Alteration in skeletal muscle lipid metabolism is thought to significantly contribute to skeletal muscle insulin resistance, which precedes and predicts type 2 diabetes (T2DM). Aerobic training is believed to protect against lipid induced insulin resistance, even in the resting state. Yet, our preliminary data found that insulin sensitivity declines earlier in aerobically trained participants than sedentary participants during modest FFA elevation, suggesting that aerobic training may promote lipid utilization when both glucose and lipid are available. Numerous studies have explained the apparent paradox of high muscle lipid content and high muscle insulin sensitivity in endurance trained humans as a consequence of reduced muscle lipid metabolites, the presumed mediator of lipid induced insulin resistance. However, these studies have only involved static measures and it remains unknown how aerobic training may alter the dynamics of muscle lipid metabolism. This proposal will study the aerobic training effect on lipid metabolism dynamics in sedentary, overweight/obese, insulin resistant humans, who may be particularly susceptible to the effects of IMCL and toxic lipid metabolites accumulation. We will test the hypothesis that aerobic training increases resting muscle lipid metabolism, as measured by markers of IMCL lipolysis, accumulation of lipid metabolites and mitochondrial utilization of fatty acids. In Aim 1, we plan t identify aerobic training effects on markers of skeletal muscle IMCL lipolysis. In Aim 2, we plan to identify aerobic training effects on the dynamics of skeletal muscle lipid metabolites. In Aim 3 we plan to identify aerobic training effects on mitochondrial utilization of fatty acids in skeleta muscle. This study is innovative in its focus on aerobic training associated alterations in skeleta muscle lipid kinetics, particularly in the use of a cutting-edge, stable isotope, pulse-chase desig to dynamically examine IMCL accumulation and lipid metabolites. By focusing on sedentary, overweight/obese, insulin resistant subjects, we are studying a population particularly susceptible to dysfunctional skeletal muscle lipid metabolism that may significantly benefit from aerobic training. The significance of this study will be to provide important insights into the mechanistic benefit of aerobic training on skeletal muscle lipid metabolism, opening new directions for treatment of insulin resistance and its consequent complications of T2DM.